Oral epithelium response of electronic cigarette users to electronic cigarette.

BACKGROUND: Electronic cigarettes are increasing in popularity, but there is only little information on their biologic effects on the oral epithelium, the initial site exposed to electronic cigarette smoke. METHODS: We assessed the oral epithelium response to electronic cigarettes by comparing the histology and RNA transcriptome (mRNA and miRNA) of healthy electronic cigarette vapers to nonsmokers. mRNA was assessed based on: (1) genome-wide; (2) genes previously identified as dysregulated in the oral epithelium of electronic cigarette vapers versus nonsmokers; (3) immune and inflammatory-related genes previously identified as dysregulated in the nasal epithelium of electronic cigarette vapers compared to nonsmokers; (4) genes previously identified as dysregulated in the small airway epithelium of nonsmokers following an acute exposure to electronic cigarette; and (5) genes related to the initial steps of COVID-19 infection. In addition, miRNA was assessed genome-wide. Comparisons were performed using analysis of variance, and Benajmini-Hochberg corrected p < 0.05 was considered significant. RESULTS: The histology of the epithelium, lamina propria and basal layer in electronic cigarette vapers appeared normal. Assessment of mRNA and miRNA, based on all gene lists, did not identify any genes significantly modified in the oral epithelium of electronic cigarette vapers in response to electronic cigarette use. CONCLUSION: An average history of 2 years of vaping results in no detectable histologic or transcriptome abnormalities in the buccal mucosa.

CREB-dependent LPA-induced signaling initiates a pro-fibrotic feedback loop between small airway basal cells and fibroblasts.

BACKGROUND: Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation. METHODS: To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed. RESULTS: LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF. CONCLUSION: Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.

Expression of the SARS-CoV-2 ACE2 Receptor in the Human Airway Epithelium.

Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium.Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium.Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection-related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2-related microRNA.Measurements and Main Results:1) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3) ACE2 is upregulated in the SAE by smoking, significantly in men; 4) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5) ACE2 is expressed in airway epithelium differentiated in vitro on air-liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers.Conclusions:ACE2, the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.

Cell-specific expression of lung disease risk-related genes in the human small airway epithelium.

BACKGROUND: The human small airway epithelium (SAE) plays a central role in the early events in the pathogenesis of most inherited and acquired lung disorders. Little is known about the molecular phenotypes of the specific cell populations comprising the SAE in humans, and the contribution of SAE specific cell populations to the risk for lung diseases. METHODS: Drop-seq single-cell RNA-sequencing was used to characterize the transcriptome of single cells from human SAE of nonsmokers and smokers by bronchoscopic brushing. RESULTS: Eleven distinct cell populations were identified, including major and rare epithelial cells, and immune/inflammatory cells. There was cell type-specific expression of genes relevant to the risk of the inherited pulmonary disorders, genes associated with risk of chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis and (non-mutated) driver genes for lung cancers. Cigarette smoking significantly altered the cell type-specific transcriptomes and disease risk-related genes. CONCLUSIONS: This data provides new insights into the possible contribution of specific lung cells to the pathogenesis of lung disorders.

Role of KRAS in regulating normal human airway basal cell differentiation.

BACKGROUND: KRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation. METHODS: To assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo. RESULTS: siRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers. CONCLUSIONS: Together, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

Characterization of an immortalized human small airway basal stem/progenitor cell line with airway region-specific differentiation capacity.

BACKGROUND: The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line. METHODS: Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF. RESULTS: The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that "cilium" and "immunity" were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2-1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF. CONCLUSIONS: The immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Ontogeny and Biology of Human Small Airway Epithelial Club Cells.

RATIONALE: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi). OBJECTIVES: To define the ontogeny and biology of the human small airway epithelium club cell. METHODS: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology. MEASUREMENTS AND MAIN RESULTS: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5)+ basal cells and SCGB1A1 (secretoglobin family 1A member 1)+ club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5+ SCGB1A1- small airway epithelial basal cells differentiate into SCGB1A1+KRT5- club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses. CONCLUSIONS: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

A comparison of epithelial-to-mesenchymal transition and re-epithelialization.

Wound healing and cancer metastasis share a common starting point, namely, a change in the phenotype of some cells from stationary to motile. The term, epithelial-to-mesenchymal transition (EMT) describes the changes in molecular biology and cellular physiology that allow a cell to transition from a sedentary cell to a motile cell, a process that is relevant not only for cancer and regeneration, but also for normal development of multicellular organisms. The present review compares the similarities and differences in cellular response at the molecular level as tumor cells enter EMT or as keratinocytes begin the process of re-epithelialization of a wound. Looking toward clinical interventions that might modulate these processes, the mechanisms and outcomes of current and potential therapies are reviewed for both anti-cancer and pro-wound healing treatments related to the pathways that are central to EMT. Taken together, the comparison of re-epithelialization and tumor EMT serves as a starting point for the development of therapies that can selectively modulate different forms of EMT.

Predicted deleterious variants in the human genome relevant to gene therapy with adeno-associated virus vectors.

Based on the observation that humans have variable responses of gene expression with the same dose of an adeno-associated vector, we hypothesized that there are deleterious variants in genes coding for processes required for adeno-associated virus (AAV)-mediated gene transfer/expression that may hamper or enhance the effectiveness of AAV-mediated gene therapy. To assess this hypothesis, we evaluated 69,442 whole genome sequences from three populations (European, African/African American, and Qatari) for predicted deleterious variants in 62 genes known to play a role in AAV-mediated gene transfer/expression. The analysis identified 5,564 potentially deleterious mutations of which 27 were classified as common based on an allele frequency ≥1% in at least one population studied. Many of these deleterious variants are predicated to prevent while others enhance effective AAV gene transfer/expression, and several are linked to known hereditary disorders. The data support the hypothesis that, like other drugs, human genetic variability contributes to the person-to-person effectiveness of AAV gene therapy and the screening for genetic variability should be considered as part of future clinical trials.

Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids.

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3ß-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

Smoking shifts human small airway epithelium club cells toward a lesser differentiated population.

The club cell, a small airway epithelial (SAE) cell, plays a central role in human lung host defense. We hypothesized that subpopulations of club cells with distinct functions may exist. The SAE of healthy nonsmokers and healthy cigarette smokers were evaluated by single-cell RNA sequencing, and unsupervised clustering revealed subpopulations of SCGCB1A1+KRT5loMUC5AC- club cells. Club cell heterogeneity was supported by evaluations of SAE tissue sections, brushed SAE cells, and in vitro air-liquid interface cultures. Three subpopulations included: (1) progenitor; (2) proliferating; and (3) effector club cells. The progenitor club cell population expressed high levels of mitochondrial, ribosomal proteins, and KRT5 relative to other club cell populations and included a differentiation branch point leading to mucous cell production. The small proliferating population expressed high levels of cyclins and proliferation markers. The effector club cell cluster expressed genes related to host defense, xenobiotic metabolism, and barrier functions associated with club cell function. Comparison of smokers vs. nonsmokers demonstrated that smoking limited the extent of differentiation of all three subclusters and altered SAM pointed domain-containing Ets transcription factor (SPDEF)-regulated transcription in the effector cell population leading to a change in the location of the branch point for mucous cell production, a potential explanation for the concomitant reduction in effector club cells and increase in mucous cells in smokers. These observations provide insights into both the makeup of human SAE club cell subpopulations and the smoking-induced changes in club cell biology.

Sonic hedgehog expands diaphyseal trabecular bone altering bone marrow niche and lymphocyte compartment.

Bone marrow contains distinct microenvironments that regulate hematopoietic stem cells (HSCs). The endosteal HSC niche includes osteoblasts, mineral, and extracellular matrix proteins that interact through various molecular signals to control HSCs. Sonic hedgehog (Shh) is a morphogen involved in the regulation of skeletal development and hematopoiesis, but the effects of Shh on bone in relation to the HSC niche are not well understood. We demonstrate that systemic overexpression of Shh in mice increases osteoblast number with the resultant formation of new trabeculae in the femoral diaphysis. Suggestive of a functional change in the hematopoietic niche, numbers of Lin(-) Sca-1(+) c-Kit(+) cells with hematopoietic progenitor function expand, although cells with in vivo repopulating capacity in the wild-type environment do not increase. Instead, Shh mediates a decrease in number of bone marrow lymphocytes accompanied by a decreased expression of stromal-derived growth factor 1 (SDF-1) and a decrease in Flk2-expressing Lin(-) Sca-1(+) c-Kit(+) cells, indicating a modulation of early lymphopoiesis. This is caused by a microenvironment-induced mechanism as Shh treatment of bone marrow recipients, but not donors, results in a dramatic depletion of lymphocytes. Together, these data suggest that Shh mediates alterations in the bone marrow hematopoietic niche affecting the early lymphoid differentiation.

Single-Cell Transcriptome Analysis of Mouse Liver Cell-Specific Tropism and Transcriptional Dysregulation Following Intravenous Administration of AAVrh.10 Vectors.

Capitalizing on liver tropism of adeno-associated viral (AAV) vectors, intravenous vector administration is commonly used to genetically modify hepatocytes, a strategy currently in clinical trials for a number of liver-based hereditary disorders. Although hepatocytes are known to exhibit extensive phenotypic heterogeneity influenced by liver zonation and dietary cycle, there is little data available for the tropism capacity, as well as the potential transcriptional dysregulation, of AAV vectors for specific liver cell types. To assess these issues, we employed single-cell RNA sequencing of the mouse liver after intravenous administration of the liver tropic AAVrh.10 vector to characterize cell-specific AAV-mediated transgene expression and transcriptome dysregulation. Wild-type 8-week-old male C57Bl/6 mice under normal feed cycle were randomly divided into three groups and intravenously administered phosphate-buffered saline (PBS), AAVrh.10Null (no transgene), or AAVrh.10mCherry (marker gene). Overall, a total of 46,500 liver cells were sequenced. The single-cell transcriptomic profiles were grouped into three separate clusters of hepatocytes (Ttr-enriched "Hep1," Tat-enriched "Hep2," and Alb-enriched "Hep3") and multiple other cell types. The hepatocyte diversity was driven by glucose and lipid homeostasis signaling. Assessment of the transgene expression demonstrated that AAVrh.10 is primarily Hep1-tropic, with a 10-gene signature positively correlated with AAVrh.10-mediated transgene expression. The transgene expression was less in Hep2 and Hep3 cells with a high receptor tyrosine kinase phenotype. Importantly, AAVrh.10 vector interactions with the liver markedly altered the transcriptional patterns of all cell types, with modified genes enriched in pathways of complement and coagulation cascade, cytochrome P450, peroxisome, antigen processing and presentation, and endoplasmic reticulum protein processing. These observations provide insights into the liver cell-specific consequences of AAV-mediated liver gene transfer, far beyond the well-known organ-specific expression of the vector-delivered transgene.

Anti-Phospho-Tau Gene Therapy for Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder caused by repetitive trauma to the central nervous system (CNS) suffered by soldiers, contact sport athletes, and civilians following accident-related trauma. CTE is a CNS tauopathy, with trauma-induced inflammation leading to accumulation of hyperphosphorylated forms of the microtubule-binding protein Tau (pTau), resulting in neurofibrillary tangles and progressive loss of neurons. At present, there are no therapies to treat CTE. We hypothesized that direct CNS administration of an adeno-associated virus (AAV) vector coding for an anti-pTau antibody would generate sufficient levels of anti-pTau in the CNS to suppress pTau accumulation thus interrupting the pathogenic process. Using a serotype AAVrh.10 gene transfer vector coding for a monoclonal antibody directed against pTau, we demonstrate the feasibility of this strategy in a murine CTE model in which pTau accumulation was elicited by repeated traumatic brain injury (TBI) using a closed cortical impact procedure over 5 days. Direct delivery of AAVrh.10 expression vectors coding for either of the two different anti-pTau antibodies to the hippocampus of these TBI mice significantly reduced pTau levels across the CNS. Using doses that can be safely scaled to humans, the data demonstrate that CNS administration of AAVrh.10anti-pTau is effective, providing a new strategy to interrupt the CTE consequences of TBI.

Dysregulation of club cell biology in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic fibrotic lung disease with an irreversible decline of lung function. "Bronchiolization", characterized by ectopic appearance of airway epithelial cells in the alveolar regions, is one of the characteristic features in the IPF lung. Based on the knowledge that club cells are the major epithelial secretory cells in human small airways, and their major secretory product uteroglobin (SCGB1A1) is significantly increased in both serum and epithelial lining fluid of IPF lung, we hypothesize that human airway club cells contribute to the pathogenesis of IPF. By assessing the transcriptomes of the single cells from human lung of control donors and IPF patients, we identified two SCGB1A1+ club cell subpopulations, highly expressing MUC5B, a significant genetic risk factor strongly associated with IPF, and SCGB3A2, a marker heterogeneously expressed in the club cells, respectively. Interestingly, the cellular proportion of SCGB1A1+MUC5B+ club cells was significantly increased in IPF patients, and this club cell subpopulation highly expressed genes related to mucous production and immune cell chemotaxis. In contrast, though the cellular proportion did not change, the molecular phenotype of the SCGB1A1+SCGB3A2high club cell subpopulation was significantly altered in IPF lung, with increased expression of mucins, cytokine and extracellular matrix genes. The single cell transcriptomic analysis reveals the cellular and molecular heterogeneity of club cells, and provide novel insights into the biological functions of club cells in the pathogenesis of IPF.

Dysfunctional glycogen storage in a mouse model of alpha1-antitrypsin deficiency.

Autophagy is an intracellular pathway that contributes to the degradation and recycling of unfolded proteins. Based on the knowledge that autophagy affects glycogen metabolism and that alpha(1)-antitrypsin (AAT) deficiency is associated with an autophagic response in the liver, we hypothesized that the conformational abnormalities of the Z-AAT protein interfere with hepatocyte glycogen storage and/or metabolism. Compared with wild-type mice (WT), the Z-AAT mice had lower liver glycogen stores (P < 0.001) and abnormal activities of glycogen-related enzymes, including acid alpha-glucosidase (P < 0.05) and the total glycogen synthase (P < 0.05). As metabolic consequences, PiZ mice demonstrated lower blood glucose levels (P < 0.05), lower body weights (P < 0.001), and lower fat pad weights (P < 0.001) compared with WT. After the stress of fasting or partial hepatectomy, PiZ mice had further reduced liver glycogen and lower blood glucose levels (both P < 0.05 compared WT). Finally, PiZ mice exhibited decreased survival after partial hepatectomy (P < 0.01 compared with WT), but this was normalized with postoperative dextrose supplementation. In conclusion, these observations are consistent with the general concept that abnormal protein conformation and degradation affects other cellular functions, suggesting that diseases in the liver might benefit from metabolic compensation if glycogen metabolism is affected.

Modification of gene expression of the small airway epithelium in response to cigarette smoking.

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.

Up-regulation of expression of the ubiquitin carboxyl-terminal hydrolase L1 gene in human airway epithelium of cigarette smokers.

Neuroendocrine differentiation is a common feature of lung cancer and increased numbers of neuroendocrine cells and their peptides have been described in chronic smokers. To understand the effects of cigarette smoking on the gene expression profile of neuroendocrine cells, microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals [normal nonsmokers, normal smokers, smokers with early chronic obstructive lung disease (COPD), and smokers with established COPD]. Of 11 genes considered to be neuroendocrine cell specific, only ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a member of the ubiquitin proteasome pathway, was consistently up-regulated in smokers compared with nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemical analysis of bronchial biopsies of smokers compared with nonsmokers. UCHL1 expression was evident only in neuroendocrine cells of the airway epithelium in nonsmokers; however, UCHL1 was also expressed in ciliated epithelial cells in smokers. This observation may add further weight to recent observations that ciliated cells are capable of transdifferentiating to other airway epithelial cells. In the context that UCHL1 is involved in the degradation of unwanted, misfolded, or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.

Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH-/-). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.